In situ heat induced antigen recovery and staining method

ABSTRACT

An automated in situ heat induced antigen recovery and staining method and apparatus for treating a plurality of microscope slides. The process of heat induced antigen recovery and the process of staining the biological sample on the microscope slide are conducted in the same apparatus, wherein the microscope slides do not need to be physically removed from one apparatus to another. The reaction conditions for treating a slide can preferably be controlled independently, including the individualized application of reagents to each slide and the individualized treatment of each slide.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 14/072,481,filed Nov. 5, 2013, which is a continuation of U.S. Ser. No. 13/291,521,filed Nov. 8, 2011, which is a continuation of U.S. Ser. No. 12/495,152,filed Jun. 30, 2009, now U.S. Pat. No. 8,052,927, which is acontinuation of U.S. Ser. No. 11/807,841, filed May 30, 2007, nowabandoned, which is a continuation of U.S. Ser. No. 10/245,035, filedSep. 13, 2002, now U.S. Pat. No. 7,250,301, which is a divisional ofU.S. Ser. No. 09/612,605, filed Jul. 7, 2000, now U.S. Pat. No.6,534,008, which claims the benefit of U.S. Provisional Application Ser.No. 60/142,789, filed Jul. 8, 1999, each of which is hereby incorporatedby reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

BACKGROUND

The present invention is related to the field of treating samples onmicroscope slides and more specifically to the field of heat inducedantigen recovery and staining.

Antigen recovery, also known as antigen unmasking, antigen epitopeunmasking, antigen retrieval or heat induced epitope recovery (HIER) isa process in which biological samples (e.g., cells, tissues, blood,fluids) are treated under heat with a series of aqueous or non-aqueousreagents and buffers (e.g., citrate, EDTA, and urea) for the purpose ofexposing the presence of specific types of antigens or biochemicalfeatures in the biological samples. HIER is regarded as a pre-treatmentprocedure to be performed prior to the beginning of a specific stainingprotocol to identify cellular components.

Biological samples must be preserved after removal from the body. Thispreservation process, known as fixation, kills and localizes thebiological material. One of the most common fixatives used widely in thepreservation of biological materials is formalin, a 10% aqueous solutionof formaldehyde. This fixative, along with other widely utilizedfixatives, produces a cross-linking network around specific sites in thebiological material. These sites are known as antigens, and during thefixation process become “masked,” by the fixative and thus “invisible”to detection by certain stains. HIER is used as a pre-treatment processto “unmask,” “retrieve” or “recover.” This process is usually conductedon formalin fixed paraffin embedded tissue sections or cellularpreparations mounted on microscope slides.

U.S. Pat. No. 5,244,787 teaches a process of antigen retrieval whereinone or more slides are placed in an aqueous solution within a microwaveoven and heated to boiling or near-boiling temperatures. These slidesare all treated together in a rack that has been placed in a bath of thesolution. The slides are near boiling temperatures for 5-30 minutes,generally around 10 minutes. Due to excessive evaporation from the bath,the patent teaches that the solution should not drop below thebiological sample on the slide because drying out of the sample isdeleterious. This process further teaches that after boiling ornear-boiling for several minutes, usually 5 minutes, one may have to addmore solution to the container to prevent the solution from excessiveevaporation and subsequent exposure of the samples on the slides. Afterthe addition of more liquid, the process is continued until the desiredtime is completed. The disclosure of U.S. Pat. No. 5,244,787 is limitedto the use of a microwave oven as the source of heating. More recentadvances, which have been published, include the use of different typesof heating devices such as electric pressure cookers, electric steamers,electric conduction heating surfaces utilizing pressure cookers,steamers, and also steam driving autoclaves (J. of Pathology,179:347-352, 1996; Biotechnic & Histochemistry, 71(5):263-270, 1996;Biotechnic & Histochemistry, 71(4):190-195, 1996; J. of Histochemistry &Cytochemistry, 45(3): 327-342, 1997).

Although these published methodologies treat the biological sample withdifferent types of solutions and with varying types of chemicals and atdifferent pH's, all teach that all slides are treated together in a bathof the heated solution. After the slides have cooled for a period oftime, they are removed from the heating device and they are transferredto another apparatus where they are manually or automatically stainedusing various reagents. This pre-treatment process of heating andremoving the slides from the heating device for staining in a separatedapparatus is highly cumbersome and inefficient. The only automated HIERor antigen retrieval instrument available is the BIOGENEX i1000. Thisinstrument, however, still employs the use of the known technology oftreating the slides as a group in a container filled with heatedsolutions. A technician must still remove the slides from the antigenretrieval (heating) instrument and place them in an automated stainerinstrument to complete the required staining protocol.

As noted herein, no currently available automated or semi-automatedstaining instruments specifically teach the ability to heat an aqueousor non-aqueous liquid for the unmasking of antigens. The instrumentsthat do automated or semi-automated staining limit their scope to thattask alone, and don't address the task of HIER or antigen retrievalpre-treatments. U.S. Pat. Nos. 5,073,504 and 4,847,208 teach use of achamber for enclosing and staining a microscope slide but neitherteaches use of a heating device to boil a liquid and the user must addthe primary antibody manually through a hinged door on top of thechamber. U.S. Pat. Nos. 4,777,020; 4,798,706; and 4,801,431 teach use ofa vertical staining “capillary gap” methodology wherein two specialslides placed front to front causing an air gap through which liquidsare drawn by capillary movement. This gap can only hold a small volume(approx. 300 microliters) of liquid. If heated to near boilingconditions the liquid would evaporate through all four open sides,immediately causing the biological sample to dry. This end result istrue also for another instrument, shown in U.S. Pat. No. 5,804,141. U.S.Pat. Nos. 5,595,707; 5,654,200; 5,654,199; and 5,650,327 teach reducingevaporative loss by utilizing an oil layer on top of the aqueous layer.This is somewhat effective in reducing the amount of evaporative loss at37° C. but the volume of the aqueous layer (approx. 300 microliters) isagain minimal, and if heated to boiling, would cause the aqueous layerto dry out leaving only the oil layer present thus damaging thebiological sample unless more aqueous reagent was applied during thetreatment process. U.S. Pat. No. 5,425,918 also teaches use of smallamounts of liquids that are sprayed on the slide and can only heat theslide to 37° C. U.S. Pat. Nos. 5,645,144 and 5,947,167 teach use of anopen top chamber present around the slide and use a non-rotating coverabove the slides to reduce evaporation. There is no teaching of hightemperature heating of a liquid for a substantial amount of time.Further, even if one would increase the temperature of the slide, thenon-rotating top of the chamber would allow so much evaporative lossthat the solution would never reach boiling or near boilingtemperatures, nor would it maintain the boiling conditions for 10minutes or longer. U.S. Pat. No. 5,645,114 teaches use of small volumesof liquids (up to 500 microliters) and has no ability to stopevaporative loss if the slide temperature reaches boiling conditions.

As a result, none of these systems could hold sufficient liquid on topof a slide (e.g., 4 ml) and are enclosed in a chamber which is properlyvented to minimize the energy loss from evaporation to cause sufficientheating to boil the liquid on the slide for the length of time generallyrequired to cause antigen unmasking (e.g., 10-30 minutes).

There remains a need for an apparatus which can perform the task of HIERwith subsequent staining treatment without the need of switching theslides from one apparatus to another and wherein the treatment of allmicroscope slides can occur simultaneously thereby increasingefficiency. Of the automated stainers available today, there is not oneinstrument that has the ability to overcome the inherent problems ofheating an aqueous or non-aqueous solution at a sufficient volumewithout the undesirable effect of evaporative heat loss and subsequentvolume decrease of the solution. The negative effects of evaporation aresignificant. The ability of a liquid to reach boiling or near boilingtemperature on a microscope slide is dependent on the containment andcontrol of the steam or vapor generated during the heating process. Itis the object of the invention contemplated herein to provide acompletely automated HIER apparatus which can recover antigens withmultiple types of recovery buffers simultaneously, each specific to itsrespective microscope slide and which can also be used to stain themicroscope slides as well.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an apparatus of the invention (shownwithout a pressing element for crushing a reagent capsule).

FIG. 2 is a cross-sectional view of the apparatus of FIG. 1 (shown witha pressing element for crushing a reagent capsule).

FIG. 3A is a cross-sectional view of the apparatus of FIG. 1 (shown witha reaction compartment having a raised slide support surface) takenthrough line 3A-3A.

FIG. 3B is a cross-sectional view of the apparatus of FIG. 1 (shown witha reaction compartment having a lowered slide support surface) takenthrough line 3B-3B.

FIG. 4 is a cross-sectional view of an alternative embodiment of theapparatus of the present invention having an alternate type of slidesupport surface.

FIG. 5 is a perspective view of a reagent strip of the presentinvention.

FIG. 6 is a cross-sectional view of the reagent strip of FIG. 5 takenthrough the line 6-6.

FIG. 7 is an elevational view of a modular apparatus containing aplurality of the apparatus of FIG. 1.

FIG. 8 is a flow chart showing a preferred sequence of steps in themethod of the present invention.

FIG. 9 is a schematic view of an apparatus of the invention and amicroprocessor which controls the apparatus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to an automated method and apparatusfor treating biological samples on microscope slides for unmasking(“retrieving” or “recovering”) epitopes or antigens of the biologicalsamples and then staining or otherwise treating the biological samples.The automated apparatus comprises an array of individual reactioncompartments, each of which is used to treat a single microscope slide(also referred to herein as a “slide”), wherein each reactioncompartment preferably can function and can be controlled independentlyof the other reaction compartments in the array. Each reactioncompartment in the array comprises a support element comprising asurface upon which a microscope slide can be supported and positionedadjacent or inserted into the compartment for treatment with a reagent.The support element further comprises, in a preferred embodiment, aconduction type heating element for heating the microscope slide to apredetermined treatment temperature when desired. The support elementwith the microscope slide thereon can be raised into or adjacent thereaction compartment for treatment of the microscope slide, or loweredor removed from the reaction compartment for placement of a microscopeslide onto or removed from the support surface or for removal of areagent or rinsing solution from the microscope slide during thetreatment process.

Reagents, such as antibodies, enzymes, rinse buffers, antigen recoverybuffers, or stains, are contained in an individualized reagentdispensing strip which is specific for each microscope slide to betreated. Since each microscope slide and reaction compartment isgenerally provided with its own reagent dispensing strip, eachmicroscope slide can be treated independently with a different set ofreagents (a particular treatment protocol) while being treatedsimultaneously with other microscope slides. Similarly, in an especiallypreferred embodiment of the invention, each microscope slide can beheated separately, as well as treated with a different treatmentprotocol. The apparatus of the present invention therefore comprises, ina preferred embodiment, a plurality of individualized reactioncompartments in a chamber which can be substantially closed forminimizing evaporation during heating. A microscope slide can besupported in each reaction compartment, and each microscope slide can beheated separately therein. A reagent dispensing strip containing aplurality of individually contained reagents (reagent “bubbles”,“blisters” or “capsules”) is positioned upon an upper portion of eachreaction compartment, and at an appropriate time, a reagent from eachreagent dispensing strip is expelled from a reagent capsule undercompression and is thereby applied to the biological sample on themicroscope slide. Or, a reagent, such as an antigen recovery buffer canbe introduced via a separate dispenser. The term “reagent” is definedherein to include any type of fluid material that may be applied to thebiological material on the microscope slide, including antibodies,stains, enzymes, buffers, rinses, or washes, or any other materialapplied in the process of antigen recovery or treating the biologicalmaterial on the microscope slide to be viewed under the microscope.

During an antigen recovery step, the microscope slide, sample, andantigen recovery buffer thereon are heated to an appropriate temperaturefor a predetermined duration to cause the antigen recovery buffer toreact with the sample on the microscope slide, after which the antigenrecovery buffer is removed from the microscope slide, preferably bywashing or flooding the microscope slide or chamber containing themicroscope slide with a rinse buffer and allowing the rinse buffer todrain off by gravity or by blowing the solution off the microscope slideusing pressurized air. Each microscope slide may be treated in the samemanner, or may be treated with different reagents using a differenttreatment protocol, preferably simultaneously, yet independently.

When a reagent is provided via a reagent dispensing strip, the apparatusis preferably equipped with a drive mechanism for causing the reagentdispensing strip to be advanced in a forward direction wherein eachreagent capsule in succession is positioned above an aperture in thecompartment through which the reagent in the capsule is delivered. Thereagent dispensing strip may be advanced using rollers positioned alongthe upper end of the compartment or a pushing mechanism which pushesupon the rear end of the reagent dispensing strip. The reagent in thereagent capsule of the reagent dispensing strip is to be applied to themicroscope slide by a pressing mechanism which, in a preferred version,compresses and thereby crushes the reagent capsule and causes thereagent to be expelled and deposited directly onto the microscope slide.

In a preferred method of the present invention, a plurality ofmicroscope slides, each having thereon a sample to be treated, isprovided. Each microscope slide is positioned upon a support elementwhich is then moved into an application position. A plurality of reagentdispensing strips is provided, one for each microscope slide to betreated. Each microscope slide is subjected to an antigen recovery stepthen is treated by applying a reagent from its corresponding reagentdispensing strip. Each microscope slide can be handled differently, ifdesired, during the treatment cycle. After a predetermined duration, themicroscope slide and support element is moved to a removal positionwherein the reagent is removed, preferably in between reagentapplications, by treatment with a rinsing solution to remove the reagentprior to further treatment. Each microscope slide can be treatedaccording to the treatment protocol specific to that sample or thatparticular microscope slide. All microscope slides may be treated usingthe same protocol, or one or more, or all, of the microscope slides maybe treated using a different protocol.

An example of a treatment protocol comprises:

-   -   1) antigen recovery, 10 minutes at 98° C.,    -   2) cool, 20 minutes,    -   3) rinse buffer,    -   4) primary antibody, 30 minutes,    -   5) rinse,    -   6) biotinylated linking antibody, 10 minutes,    -   7) rinse buffer,    -   8) peroxidase labeled streptavidin label,    -   9) rinse buffer,    -   10) 3,3′-diaminobenzidine chromogen,    -   11) rinse buffer,    -   12) chromogen enhancer,    -   13) rinse buffer, and    -   14) counter stain.

A variety of other treatment protocols are well known to those ofordinary skill in the art and further discussion of them herein is notdeemed necessary. Each microscope slide, if necessary, may be heatedprior to application of the reagent, if necessary, then may be cooled asthe reagent is removed, then reheated, if necessary, prior to or afteraddition of the next reagent. The entire process is run automaticallyonce the microscope slide is disposed onto the support element, and thereagent dispensing strip is positioned upon the upper side of thereaction compartment.

Turning now to the drawings, a specific embodiment of the apparatus ofthe present invention is shown in FIGS. 1-6. Although FIGS. 1-6 show apreferred version of the invention, it will be understood that theembodiment shown in FIGS. 1-6 is but one of many possible versions ofthe apparatus enabled herein which will come to the mind of a person ofordinary skill in the art.

Shown in FIG. 1, and designated therein by the general reference numeral10 is an antigen recovery and staining apparatus constructed inaccordance with the present invention. The antigen recovery and stainingapparatus 10 comprises a treatment chamber 12 which further comprises aplurality of reaction compartments 14 (see FIGS. 2-4). Preferably thetreatment chamber 12 generally comprises from 10 to 20 reactioncompartments 14 but may contain more or fewer. Each reaction compartment14, when enclosed, minimizes evaporation of a reagent solution when amicroscope slide is exposed to high temperature pretreatment conditions.Each reaction compartment 14 has an upper side 16 having an opening 18therein, a lower side 20, and a pair of sidewalls 22 which extend fromthe rear end 23 a of the treatment chamber 12 to the front end 23 b ofthe treatment chamber 12. Positioned above each reaction compartment 14is a reagent dispensing strip holder 24 for holding and guiding areagent dispensing strip 26 (see FIGS. 5 and 6). Each reagent dispensingstrip 26 has a front end 28 and a rear end 30 and a plurality ofcapsules 32 made of a crushable plastic material such as polyethylene oranother suitable material (e.g., polypropylene or polystyrene) and whichmay include one or more multiple capsules 32 a. The size of each capsule32 or multiple capsule 32 a may be adjusted to accommodate the amount ofreagent which is desired to be applied to a microscope slide 44. Eachcapsule 32 or multiple capsule 32 a contains a reagent or treatmentsolution which is intended to be applied to a biological material on themicroscope slide 44. Multiple capsule 32 a is useful in a method whereintwo or more reagents must be contained separately before being appliedto the microscope slide 44. When the multiple capsule 32 a is crushed bythe pressing mechanism 36, two or more reagents contained within thecapsule 32 a are combined and simultaneously applied to the microscopeslide 44.

Other embodiments of the reagent dispensing strip 26 and the reagentcapsule 32 and multiple capsule 32 a will readily be apparent to one ofordinarily skill in the art. For example, each reagent dispensing strip26 may comprise a one or more “blank” spaces for insertion ofindividualized capsules 32 by a user. Below each capsule 32 or multiplecapsule 32 a is an aperture or weak area 34 in the reagent dispensingstrip 26 through which the reagent in the capsule 32 or multiplecapsules 32 can be forced by a pressing mechanism 36. The “blank” spaceor space left by the puncturing of a capsule 32 or 32 a, or vents in thereagent dispensing strip 26 may function to release pressure, steam orvapors produced during the treatment process. The reagent dispensingstrip 26 is advanced in a direction 37 toward the front end 23 b of thetreatment chamber 12 by a reagent strip drive mechanism 38 driven, forexample, by an electric motor which in FIGS. 1, 3A and 3B is shown as apushing mechanism comprising a threaded shaft, but which may instead bya mechanism (not shown) comprising rollers which drive, draw or “pull”the reagent strip holder 24 in a forward direction 37.

Each reaction compartment 14 further comprises at its lower side 20 aslide support element 40 having a slide tray 42 upon which themicroscope slide 44 can be positioned and held for treatment. The slidesupport elements 40 together comprise a slide support assembly 39. Withthe microscope slide 44 disposed on the slide support element 40, theslide support element 40 and the microscope slide 44 are positioned inan application position to fit adjacent the lower side 20 of thereaction compartment 14, thereby constituting an openable bottom of thereaction compartment 14. The slide support element 40 further has aheating element 46 incorporated therein for heating the microscope slide44 as discussed elsewhere herein. In one embodiment, the slide supportelement 40 has a hinge 48 for enabling the slide support element 40 tobe moved (raised) into an application position (FIG. 3A) and therefromlowered (e.g., tilted) into an opened position (see FIG. 3B).Alternatively, the slide support element 40 may be raised and loweredinto position by another mechanism, such as a stepper motor 58 and screwdrive 59 mechanism (FIG. 4). Each reaction compartment 14 furthercomprises a manifold 50 which comprises, in a preferred embodiment, aplurality of reagent dispensing ports or elements including, for examplebut not limited to, an antigen recovery buffer dispenser 51 connectedvia an antigen recovery buffer supply line 51 a to an antigen recoverybuffer supply (not shown), a rinse buffer dispenser 52 connected via arinse buffer supply line 52 a to a rinse buffer supply (not shown) andan air pressure nozzle (pressurized air nozzle) 54 connected via an airline 54 a to an air supply (not shown). The antigen recovery bufferdispenser 51 applies an antigen recovery buffer to the microscope slide44 for the antigen recovery treatment step prior to staining or otherpreparation of the biological material on the microscope slide 44. Therinse buffer dispenser 52 applies a rinse buffer 56 to the microscopeslide 44 to rinse a reagent from the microscope slide 44. The airpressure nozzle (pressurized air nozzle) 54 functions to clear away arinse buffer 56 from the microscope slide 44. Dispensers 51 and 52 maybe used to dispense other reagents, and may constitute more than, orfewer than, the dispensers shown in FIGS. 2, 3A, 3B, and 4. Themicroscope slide 44 is generally disposed in a removal position forfacilitating removal of the rinse buffer 56 as shown in FIGS. 1 and 3B.Each slide support element 40, in a preferred embodiment, can be heatedor moved independently of any other slide support element 40, althoughone of ordinary skill in the art can envision that the slide supportelements 40 may be designed to operate in concert, i.e., simultaneously.Each reaction compartment 14 preferably can contain a volume of up to 15ml. The slide support element 40 and the microscope slide 44 supportedthereon may be in a horizontal position (e.g., FIGS. 1, 2, 3A and 4) orin a tilted position (e.g., FIGS. 1 and 3B).

The antigen recovery and staining apparatus 10 can be controlledautomatically wherein predetermined sequences and operations are carriedout using various electromechanical systems which are not shown butwhich are well known to those of ordinary skill in the art. For example,each of the steps of raising into a treatment position and lowering intoa removal position each of the slide support elements 40, applying anantigen recovery buffer, advancing each reagent dispensing strip 26,compressing each capsule 32 or 32 a of the reagent dispensing strip 26,heating each microscope slide 44 on the slide support surface 40,applying a rinse buffer 56 to the microscope slide 44, removing therinse buffer 56 or other reagent from the microscope slide 44, andtreating each microscope slide 44 independently can be automaticallycontrolled and programmed using programming methods and devices wellknown in the art. Because each reaction compartment 14 and slide supportelement 40 can be controlled independently, a microscope slide 44 caneven be removed or inserted even while other reaction compartments 14are in operation.

Preferably, a microprocessor 62, controls the antigen recovery andstaining apparatus 10 as shown in FIG. 9. That is, an operator programsthe microprocessor 62 with information such as which reactioncompartments 14 are to be used and to what temperature each is to beheated and at which steps, then programs the particular treatmentprotocol to be performed on the sample on each microscope slide 44 oneach slide support element 40. Variables in these protocols can includethe particular type of reagent dispensing strip 26 to be used, the timethat each reagent or treatment solution on the reagent dispensing strip26 will be allowed to react with the sample on the microscope slide 44,whether the microscope slide 44 will be heated, and if so to whattemperature and for how long, and the manner in which the microscopeslide 44 will be rinsed, for example. Other variables not listed hereinmay also be programmed.

The invention may further comprise a modular apparatus 60 comprising aplurality of antigen recovery and staining apparatuses 10 each servingas an individual module in the modular apparatus 60. The individualmodules can be “stacked” together for example, as shown in FIG. 7, ormay be oriented in any other desirable manner.

Shown in FIG. 8 is a schematic drawing which describes the preferredmethod of the present invention. In the first step, a microscope slide44 which has a sample disposed thereon is provided, and is disposed ontoa slide support element 40 which is moved into an application ortreatment position adjacent or against the reaction compartment 14. If aplurality of microscope slides 44 are supplied, each microscope slide 44is disposed on a separate microscope slide support element 40 and themicroscope slides 44 are moved independently or simultaneously into anapplication position.

Once in the application position, an antigen recovery buffer isinitially applied to the sample on the microscope slide 44. Microscopeslide 44 is then heated to a desired, predetermined temperature, forexample from about 120° C. to about 160° C. whereby the antigen recoverybuffer is heated to a temperature of from about 90° C. to 100° C., forexample. The microscope slide 44 is allowed to react with the reagentfor a predetermined length of time, for example, 10 to 30 minutes,preferably at 95°-98° C. Venting of steam may occur through small holes(not shown) in the reagent strip 26 or elsewhere in the reactioncompartment 14. It is not necessary to add additional antigen recoverybuffer during this step. After the reaction period is over, the slidesupport element 40 and the microscope slide 44 thereon are moved(lowered or dropped) to a removal position, if necessary, where theantigen recovery buffer is removed from the microscope slide 44, forexample, by applying a rinsing solution or buffer to the microscopeslide 44 or by gravity or by pressurized air. A rinse solution or buffermay be applied and removed more than once for treatment or for removalof a particular reagent before or after lowering the microscope slide 44to the removal position. It may be desirable to add rinse buffer to themicroscope slide 44 to cool the microscope slide 44 prior to loweringthe microscope slide 44 to the removal position, for example, by addingrinse buffer 56 to the antigen recovery buffer before the microscopeslide 44 is moved to the removal position. After the microscope slide 44has been treated for antigen recovery, another reagent can then beapplied for treatment of the sample on the microscope slide 44. In thisstep, the microscope slide 44 and slide support element 40 are thenreturned to the application position, a reagent is applied, and is thenremoved after the treatment period. The series of steps may be repeated.When the treatment of the sample is completed, the microscope slide 44is removed from the slide support element 40 for further treatment oranalysis apart from the antigen recovery and staining apparatus 10.

Changes may be made in the construction and the operation of the variouscomponents, elements and assemblies described herein or in the steps orthe sequence of steps of the methods described herein without departingfrom the scope of the invention as defined in the following claims.

What is claimed is:
 1. A method of treating biological samples,comprising: disposing a plurality of microscope slides on a first slidetray and a plurality of microscope slides on a second slide tray of anautomated antigen recovery and staining apparatus, each of themicroscope slides disposed on the first slide tray and the second slidetray having a biological sample disposed thereon, the automated antigenand staining apparatus comprising a first plurality of separate heatingelements each corresponding to one of the microscope slides disposed onthe first slide tray for independently heating each of the microscopeslides disposed on the first slide tray and a second plurality ofseparate heating elements each corresponding to one of the microscopeslides disposed on the second slide tray for independently heating eachof the microscope slides disposed on the second slide tray; andpositioning the first slide tray having the first plurality ofmicroscope slides thereon into a first treatment position, wherein thebiological samples on the first plurality of microscope slides areheated with the first plurality of heating elements, while the secondslide tray is in a second treatment position wherein the biologicalsamples on the second plurality of microscope slides are being heated bythe second plurality of heating elements.
 2. The method of claim 1,wherein the positioning of the first slide tray and the second slidetray is controlled electromechanically.
 3. The method of claim 1,further comprising automatically applying reagents to the biologicalsamples disposed on the microscope slides disposed on the first slidetray and the biological samples disposed on the second slide tray via aplurality of reagent dispensers wherein the reagent dispensers are ableto dispense reagents simultaneously and independently of each other. 4.A method of treating biological samples, comprising: disposing aplurality of microscope slides on a first slide tray and a plurality ofmicroscope slides on a second slide tray of an automated antigenrecovery and staining apparatus, each of the microscope slides disposedon the first slide tray and the second slide tray having a biologicalsample disposed thereon, the automated antigen and staining apparatuscomprising a first plurality of separate heating elements eachcorresponding to one of the microscope slides disposed on the firstslide tray for independently heating each of the microscope slidesdisposed on the first slide tray and a second plurality of separateheating elements each corresponding to one of the microscope slidesdisposed on the second slide tray for independently heating each of themicroscope slides disposed on the second slide tray; and positioning thefirst slide tray having the first plurality of microscope slides thereonfrom a first treatment position, wherein the biological samples on thefirst plurality of microscope slides are heated with the first pluralityof heating elements, while the second slide tray is in a secondtreatment position wherein the biological samples on the secondplurality of microscope slides are being heated by the second pluralityof heating elements
 5. The method of claim 4, wherein the positioning ofthe first slide tray and the second slide tray is controlledelectromechanically.
 6. The method of claim 4, further comprisingautomatically applying reagents to the biological samples disposed onthe microscope slides disposed on the first slide tray and thebiological samples disposed on the second slide tray via a plurality ofreagent dispensers wherein the reagent dispensers are able to dispensereagents simultaneously and independently of each other.